Abstract: Recent advances in genomics, proteomics, and metabolomics make the "omics" technologies powerful discovery-based tools for identifying candidate biomarkers for human diseases;however, it has not been successful so far to establish new biomarkers for clinical practice by utilizing these technologies. The main bottleneck lies in the lack of effective tools for high-throughput validation. To overcome this bottleneck I propose to develop a novel "reagent-free" mass spectrometry-based universal multiplex assay system that will provide high throughput quantitative measurements for hundreds of low-abundance protein and metabolite analytes, independent of antibody-based reagents. The goal for this technology platform is to achieve a profound advance over current MS-platforms by providing >1000-fold enhancement in analyte signal intensities, sufficient for detecting low-abundance species, and >5000-fold improvement in resolving power for extremely high specificity detection. These advances will be achieved by developing and integrating 1) a novel subambient pressure ionization source with nanoelectrospray array, 2) advanced ion-funnel interfaces, 3) novel multi-stage gas-phase ion mobility technology (differential mobility analyzer coupled to field asymmetric ion mobility spectrometry) for separating and selecting analytes of interest, and (4) a new triple-stage pentaquardrupole (QqQqQ) mass spectrometer for further isolating as well as detecting ions. The optimized platform will have a potential analytical throughput of >100 samples per day, sensitivity for broadly analyzing low-abundance candidate biomarkers without enrichment, and the multiplexing power to monitor up to 1000 analytes simultaneously. At least 3-4 orders of magnitude enhancement in sensitivity or detection dynamic range (i.e., to a level comparable or superior to current ELISA) is anticipated, a significant advance over current assay platforms. Such a novel assay system is a disruptive technology that will revolutionize many areas of biomedical research, current medical practice, and the future of health care, as well as the biomedical research field in general through instrument commercialization. Public Health Relevance: The objective of this project is to develop a novel mass spectrometry-based universal multiplex assay system for high throughput clinical discovery and validations of candidate biomarkers of human diseases. The development will have major clinical and translational impact on current biomedical research by accelerating biomarker discovery and implementation in clinical practices. Large number of candidate biomarkers for various human diseases can be effectively screened for validation using the new assay system to discover markers for improved disease diagnosis, prognosis, and treatment.